Methods of preparing lymphocytes that express interleukin-2 and their use in the treatment of cancer

ABSTRACT

The invention provides methods of preparing autologous T-lymphocytes for re-introduction into a patient having cancer, which method comprises obtaining peripheral blood mononuclear cells (PBMCs) from a patient immunized with an antigen of the cancer, stimulating the PBMCs with the antigen of the cancer in vitro, transducing the PBMCs with a retroviral vector, which (a) comprises and expresses a human interleukin-2 (IL-2) coding sequence operably linked to a retroviral promoter, (b) does not comprise an exogenously introduced gene that enables phenotypic selection, and (c) comprises a viral envelope that efficiently transduces CD8+ T-lymphocytes; compositions comprising cells obtained in accordance with such methods; and methods of treating a patient having cancer by administering to the patient cells obtained in accordance with such methods or compositions comprising same.

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 5,000 Byte ASCII (Text) file named“234872sequence.2.TXT,” created on Aug. 20, 2007.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods of preparing autologousT-lymphocytes and tumor-infiltrating lymphocytes that expressinterleukin-2 (IL-2) and related compositions and methods of use in thetreatment of cancer.

BACKGROUND OF THE INVENTION

One major obstacle limiting the efficacy of adoptive T-cell transfer(adoptive immunotherapy) in the treatment of cancer patients is theshort-term survival of the transferred cells. In-vitro-activated T-cellsundergo apoptosis upon transfer in vivo. Accordingly, there remains aneed for improved T-cells for adoptive immunotherapy. It is an object ofthe present invention to provide such T-cells. This and other objectsand advantages of the present invention, as well as additional inventivefeatures, will be apparent from the detailed description providedherein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of preparing autologousT-lymphocytes for re-introduction into a patient having cancer. Themethod comprises:

-   -   (i) obtaining peripheral blood mononuclear cells (PBMCs) from a        patient immunized with an antigen of the cancer,    -   (ii) stimulating the PBMCs with the antigen of the cancer in        vitro, and    -   (iii) transducing the PBMCs with a retroviral vector, which (a)        comprises and expresses a human IL-2 coding sequence operably        linked to a retroviral promoter, (b) does not comprise an        exogenously introduced gene that enables phenotypic selection,        and (c) comprises a viral envelope that efficiently transduces        CD8+ T-lymphocytes, whereupon autologous T-lymphocytes are        prepared for re-introduction into a patient having cancer.

A composition comprising T lymphocytes obtained in accordance with theabove method is also provided. Seventy-five percent or more of theT-lymphocytes in the composition are CD8+, and the cells do not containan exogenously introduced gene that enables phenotypic selection.

In view of the foregoing, the present invention provides a method oftreating a patient having cancer. The method comprises administering tothe patient autologous T-lymphocytes, which have been prepared inaccordance with the above method, alone or in further combination withhuman IL-2 receptor α-chain, in amount(s) sufficient to treat thepatient for cancer.

The present invention further provides a method of preparing autologoustumor-infiltrating lymphocytes (TILs) for re-introduction into a patienthaving cancer. The method comprises:

-   -   (i) obtaining TILs from a patient, who has been optionally        immunized with an antigen of the cancer,    -   (ii) transducing the TILs, which have been optionally stimulated        with the antigen of the cancer in vitro, with a retroviral        vector, which (a) comprises and expresses a human IL-2 coding        sequence operably linked to a retroviral promoter, (b) does not        contain an exogenously introduced gene that enables phenotypic        selection, and (c) comprises a viral envelope that efficiently        transduces CD8+ TILs, whereupon autologous TILs are prepared for        re-introduction into a patient having cancer.

A composition comprising TILs obtained in accordance with the abovemethod is also provided. Seventy-five percent or more of the TILs areCD8+, and the cells do not contain an exogenously introduced gene thatenables phenotypic selection.

In view of the foregoing, the present invention provides another methodof treating a patient having cancer. The method comprises administeringto the patient autologous TILs, which have been prepared in accordancewith the above method, alone or in further combination with human IL-2receptor α-chain, in amount(s) sufficient to treat the patient forcancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the discovery that anti-tumorT-lymphocytes can be genetically modified to make their proliferationIL-2-independent without malignant transformation and/orimmortalization. In addition, by making the growth of the T-lymphocytesdependent on stimulation and by enabling the T-lymphocytes toself-select, the T-lymphocytes are particularly valuable in the adoptiveimmunotherapeutic treatment of cancer patients.

The present invention provides a method of preparing autologousT-lymphocytes or autologous TILs for re-introduction into a patienthaving cancer. The method comprises obtaining autologous T-lymphocytesor TILs from the patient. While any suitable source of T-lymphocytes canbe used, PBMCs are a preferred source. Suitable sources ofT-lymphocytes, such as PBMCs, and TILs can be isolated from the patientin accordance with methods known in the art.

Desirably, the patient, from whom the T-lymphocytes are obtained, hasbeen immunized with an antigen of the cancer in accordance with methodsknown in the art (see, e.g., Rosenberg et at., Nat. Med. 4:321-327(1998)). If TILs are obtained from the patient, the prior immunizationof the patient with an antigen of the cancer is optional. Preferably,the antigen of the cancer is one that induces a strong immune responseagainst the cancer in the patient. For example, if the cancer ismelanoma, preferably the patient is immunized with gp100, morepreferably amino acids 209-217 of gp100, and most preferably amino acids209-217 of gp100 with a methionine substitution at position 210,referred to herein as the 209-2M peptide (SEQ ID NO: 5). Her-2/Neu is apreferred antigen for immunization of a patient to be treated for breastcancer. If the patient has prostate cancer, preferably the patient isimmunized with prostate-specific antigen (PSA). A patient with coloncancer is preferably immunized with carcinoembryonic antigen (CEA).These antigens are exemplary and are not intended to be limiting.

Once a source of autologous T-lymphocytes has been obtained, the cells,such as PBMCs, are stimulated with the antigen of the cancer in vitro.If autologous TILs have been obtained, the in vitro stimulation of theTILs with the antigen of cancer is optional. The cells can be stimulatedin vitro with the antigen of the cancer to be treated by any suitablemanner. A preferred manner is exemplified in the Examples set forthherein. Desirably, the antigen used to stimulate the cells in vitro isthe same antigen used to immunize the patient. “Stimulate,”“stimulated,” and “stimulation” are all used to refer to the phenomenonof the T-cell receptor complex binding to a stimulus, such as a specificstimulus, e.g., a cancer antigen, or a non-specific stimulus, e.g., ananti-CD3 antibody (OKT3, for example, which is available fromOrthoclone, Ortho-biotech, Raritan, N.J.) or phytohemagglutinin (PHA),which results in cellular proliferation and the manifestation ofanti-tumor activity.

After the cells, such as PBMCs, have been stimulated in vitro with theantigen of the cancer to be treated, the cells are transduced with aretroviral vector, which comprises and expresses a human IL-2 codingsequence operably linked to a retroviral promoter, i.e., the retroviral5′ long terminal repeat (LTR) promoter, and which comprises a viralenvelope that efficiently transduces CD8+ T-lymphocytes. The codingsequence of a human IL-2 is available (see, e.g., Liu et al., J.Immunol. 167: 6356-6365 (2001)). The operable linkage of a codingsequence, such as that which encodes human IL-2, and a retroviralpromoter is within the ordinary skill in the art. See, e.g., Sambrookand Russell, Molecular Cloning, 3rd Ed., SCHL Press (2001). If it isdesired to increase the expression of IL-2, the retroviral promoter canbe replaced with a stronger promoter, such as the cytomegaloviral (CMV)promoter, such as in the context of a CMMP retroviral construct (Kleinet al., J. Exp. Med. 191: 1699-1709 (2000)).

The retroviral vector must comprise a viral envelope that efficientlytransduces CD8+ T-lymphocytes. A preferred viral envelope is that ofgibbon ape leukemia virus (GALV), which can be provided by the packagingcell line PG13. Alternatively, the retroviral vector can be packaged inthe cell line PA317.

Transduction of cells with retroviral vectors is within the skill in theart. See, e.g., Sambrook (2001), supra. The culture of PBMCs, TILs andtransduced PBMCs and TILs is also within the skill in the art. See,e.g., the Examples herein. Although IL-2-independent for their growth,IL-2-transduced PBMC self-regulate their growth by down-regulation ofthe expression of the transduced IL-2 gene. IL-2-transduced CD8+ TILscan secrete IL-2 upon tumor antigen stimulation, can proliferate in theabsence of exogenous IL-2 after the destruction of autologous tumorcells, without the help of CD4+ cells, and can self-select for theirgrowth in the absence of added IL-2.

If desired, the retroviral vector can further comprise and express ahuman IL-2 receptor α-chain (otherwise referred to as CD25 or Tac)coding sequence. The coding sequence of a human IL-2 receptor α-chain isavailable (see, e.g., Waldmann, J. of Biol. Chem. 266: 2681-2684(1991)). The coding sequence of a human IL-2 receptor α-chain can beoperably linked to a retroviral promoter as described above.Alternatively, a separate vector comprising and expressing a human IL-2receptor α-chain coding sequence can be introduced into the cell. Whileany suitable vector can be used and any suitable vector can be operablylinked to the IL-2 receptor α-chain coding sequence as appropriate forthe vector of choice, desirably the vector does not adversely affect theefficient transduction of CD8+ T-lymphocytes with the retroviral vectorcomprising and expressing a human IL-2 and does not adversely effect theexpression of IL-2 therein. If a separate vector is used to express ahuman IL-2 receptor α-chain, preferably the vector is a retroviralvector, such as GALV, and the IL-2 receptor α-chain coding sequence isoperably linked to a retroviral promoter.

In view of the foregoing, the present invention further provides acomposition comprising T-lymphocytes or TILs obtained in accordance withthe above methods. Desirably, 75% or more of the T-lymphocytes or TILsare CD8+, the cells do not contain an exogenously introduced gene thatenables phenotypic selection, and the composition is suitable forre-introduction into the patient from whom the T-lymphocytes wereobtained. As such, the composition can contain various other componentsas known in the pharmaceutical arts. See, e.g., Remington'sPharmaceutical Sciences, 17th ed., (Mack Publishing Company,Philadelphia, Pa.: 1985).

Also in view of the foregoing, the present invention provides a methodof treating a patient having cancer. The method comprises administeringto the patient autologous T-lymphocytes or TILs, which have beenprepared in accordance with the above methods. The autologousT-lymphocytes or TILs can be administered by any suitable route as knownin the art. The T-lymphocytes or TILs preferably are administeredintravenously, although the T-lymphocytes or TILs can be administeredintra-arterially. Preferably, 3×10¹¹ autologous T-lymphocytes or TILsare administered as a single dose. If necessary, a second dose can beadministered after about 6-8 weeks.

The method can further comprise the administration of human IL-2receptor α-chain, whether encoded in the vector encoding human IL-2, orencoded in a separate vector for introduction into the T-lymphocytes orTILs. When the cells are no longer stimulated by cancer antigen, such aswhen there no longer are any cancer cells present, the IL-2-transducedT-lymphocytes or TILs stop growing and eventually ere eliminated.

EXAMPLES

The following examples are intended to illustrate the present inventionand are not intended to limit its scope in any way.

The follow materials were used in the examples:

-   Ca++− Mg++−, Phenol red-free Hanks' balanced salt solution (HBSS)    (BioWhittaker)-   RPMI 1640 with L-Glutamine (BioWhittaker)-   HEPES, 1 M, pH 7.0, stock (BioWhittaker)-   2-Mercaptoethanol, 5.5×10⁻² M in D-PBS, stock (Gibco BRL)-   Penicillin G sodium (10,000 units/ml), streptomycin (10,000 mg/ml)    stock-   (BioWhittaker). Omit if patient is allergic to Penicillin.-   Gentamycin (50 mg/ml) stock (BioFluids). Omit if patient is allergic    to Gentamycin.-   Ciprofloxacin (Cipro 1% solution, Bayer). Omit if patient is    allergic to Ciprofloxacin.-   Fungizone (250 mcg/ml stock; Biofluids). Omit if patient is allergic    to Fungizone.-   Aim V serum free lymphocyte growth medium (Gibco)-   0.9% sodium chloride, USP (Baxter)-   Human Serum, type AB (Valley Biomedical)-   Human PBMCs-   Human albumin (Plasbumin-25, Bayer))-   Recombinant human IL-2 (106 CU/ml) (Cetus Oncology Div, Chiron*)-   OKT3 (Ortho-anti-CD3) (Orthoclone)-   g209-2M peptide 1.0 mg/ml stock (Multiple Peptide Systems)-   Lymphocyte separation medium (LSM) (ICN)-   96-well tissue culture plates, flat-bottom and U-bottom (Costar)-   Tissue culture flasks, vented cap, 25 and 175 cm² (Costar Corp)-   Centrifuge tubes, 15, 50 and 250 ml (Corning)-   Sampling site coupler (Baxter/Fenwal, Deerfield, Ill.)-   Solution transfer set (Baxter/Fenwal)-   Lifecell adapter set (Baxter/Fenwal)-   Interconnecting jumper tube, 8″ (Gibco)-   Solution transfer pump (Baxter/Fenwal)-   Culture bags, PL732 1 liter (Baxter/Fenwal)-   Culture bags, PL732 3 liter (Baxter/Fenwal)-   SBIL-2 retroviral vector (National. Gene Vector Laboratory).-   QuickExtract™ DNA extraction solution 1.0 (Epicentre)-   First strand cDNA Synthesis Kit (Amersham pharmacia biotech)-   STRATAGENE Absolutely RNA™ RT-PCR Miniprep Kit (STRATAGENE)    *Note: 50 Cetus units (CU)=300 International units (IU)

Example 1

This example describes the construction of a retroviral vectorcomprising and expressing a human IL-2 coding sequence.

The retroviral plasmid IL-2-IRES-eGFP (see, e.g., Liu et al., J.Immunol. 167: 6356-6365 (2001), where eGFP replaces YFP) was used toprepare the retroviral backbone (SB) by digestion with Bam HI, followedby blunt-ending with Klenow fragment and digestion with Not I. The IL-2cDNA insert vias also prepared from IL-2-IRES-eGFP by digestion with SalI, followed by blunt-ending with Klenow fragment and digestion with NotI. The resultant insert was directionally cloned into SB. The resultantSBIL-2 vector did not contain any other genes as confirmed by nucleotidesequencing analyses. The construct was then pseudotyped in the packagingcell line PG13, which provided Gibbon Ape Leukemia Virus (GALV) envelopeprotein. A stable PG13SBIL-2 producer clone was established and genomicanalysis revealed that this producer clone contained three copies of theintegrated retroviral IL-2 DNA. The vector supernatant produced by thisproducer clone was shown to be biologically active in transducing ahuman, non-IL-2-producing, melanoma cell line to produce IL-2 asdetected by ELISA.

Example 2

This example describes the generation of gp100-specific,melanoma-reactive, clonal lymphocytes retrovirally transduced with anexogenous IL-2 gene.

Generation of Bulk Cultures:

Complete medium (CM) consists of RPMI 1640 with 25 mM HEPES, pH 7.0, 1mM Glutamine, 50 U/ml penicillin, 50 micrograms/ml streptomycin, 2×10⁻⁵M 2-mercaptoethanol, and 10% human AB serum.

PBMCs from patients are obtained 34 weeks after 209-2M immunization byleukopheresis. PBMCs are enriched by centrifugation on lymphocytesmedium (LSM), washed two times with HBSS and cryopreserved at 1×10⁸cells/vial in one ml of human serum (Biowhittaker) with 10% dimethylsulfoxide (DMSO). PBMCs from normal donors are prepared in a similarfashion.

On day 0, one vial of PBMCs is thawed by warming rapidly to 37° C. Cellsare transferred directly into CM. PBMCs are washed twice with CM, and analiquot is counted. PBMCs (1.5×10⁶/ml) are plated in each of 4 wells ofa 24-well tissue culture plate in 2 ml of CM. 209-2M peptide is diluted1:1,000 from stock to a final concentration of 1.0 μg/ml (approximately1.0 micromolar). Plates are incubated at 37° C. in 5% CO₂.

On day 1, IL-2 is added to each well to 50 CU/ml final concentration.

On day 4, 1 ml of supernatant is replaced with 1 ml of CM plus 300 IU/mlIL-2. If cell density increases above 2×10⁶ cells/ml, cells are split tohalf of the density and each new well is fed with 1 ml of CM plus 50CU/ml IL-2.

Transduction with SBIL-2 Retroviral Supernatant:

On day 6, 2×10⁶ cells from each well are transduced with 8 mls of SBIL-2retroviral supernatant in Retronectin-coated wells of a 6-well tissueculture plate. Retronectin is coated as follows: 2 mls of 1×PBScontaining 50 μg/ml Retronectin is placed in each well of the 6-wellplate at 4° C. overnight or at room temperature for 2 hours. Wells areblocked with 2 mls of 1% BSA in 1×PBS at room temperature for 30 minutesand washed once with 2 mls of 1×PBS containing 2.5% HEPES, pH 7.0 (v/v).PBMCs/TILs (2×10⁶) cell pellet is resuspended with 8 mls of the SBIL-2retroviral supernatant, and this cell-virus mixture is applied to theRetronectin-coated plate. Cells are exposed to retroviral vector at 37°C. for 6 hours, harvested by centrifugation at 1,000×g for 10 minutes at4° C., and resuspended again in 8 mls of fresh retroviral supernatantand left in the original Retronectin-coated wells overnight at 37° C.The same transduction procedure is repeated twice on day 7 (total of 4retroviral exposures in 2 consecutive days). At the end of transduction,cells are washed with CM and resuspended in a cell density of 0.5×10⁶/mlin CM supplemented with 50 CU/ml IL-2.

On day 9, an aliquot of cells is removed from each bulk transducedculture well and assayed for activity. Briefly, 1×10⁵ PBMCs are platedper well of a flat-bottom 96-well tissue culture plate with 1×10⁵ targetcells. Typically, six different target cells are tested: T2 cells pulsedwith 1.0 μM G280, T2 pulsed with 1.0 μM G209, two HLA-mismatchedmelanoma cell lines, and two HLA-A2-matched melanoma cell lines. After24 hours of incubation, supernatants are harvested and IFN-γ isquantified by ELISA capture assay. The well (or wells), which exhibitsthe most peptide specificity and anti-tumor activity, is selected forcloning.

Cloning from Bulk Cultures:

Active bulk cultures are cloned by limiting dilution in 96-well U-bottomplates. Briefly, allogeneic PBMCs are prepared. PBMCs are obtained bythawing frozen leukopheresis vials from normal donors as describedabove. PBMCs are thawed directly into CM, washed twice, resuspended inCM, and then irradiated (34 Gy, Nordion gammacell 1000 Cs137irradiator). Enough cloning reagents for 25 plates are mixed together:500 ml of CM, 1.25×10⁸ PBMCs, 30 ng/ml OKT3, and 50 CU/ml IL-2.Responder CTL for cloning are prepared by removing an aliquot of PBMCsfrom the most active bulk culture well, counting the cell number,washing the cells in CM, and resuspending. To 100 ml of the cloningreagent mixture are added 1,000 viable responder cells from bulkculture. These are mixed well and plated in 5 U-bottom plates (2 viablecells/well) using a repeating multichannel pipette. To the remaining 400ml of cloning reagent mixture are added 1,200 viable cells. These aremixed well and plated in 20 U-bottom plates (0.6 viable cells/well)using a repeating multichannel pipette.

The final components are listed below:

Component per well Viable cells 0.6 or 2.0 Allogeneic PBMCs 5 × 10⁴ OKT3 30 ng/ml IL-2  50 CU/ml CM 200 μl

Seven days after plating, 10 CU/well (50 CU/ml) of IL-2 are added toeach well.

Fourteen days after plating, wells are screened visually for clonalgrowth. Growth-positive wells are resuspended by pipetting, and theentire contents of each well is re-plated in a well of a flat-bottom96-well plate.

Aliquots of all growth-positive wells are tested by coculture assay forspecificity and activity. Aliquots (40 μl) of cells from each well arereplated in duplicate wells of a 96-well flat-bottom plate. Target cells(1×10⁵) are added to each well. Typically, one well receives T2 pulsedwith G209 and the other well receives. T2 pulsed with G280. Alternately,one well receives an HLA-A2 matched tumor and the other well receives anHLA mismatched tumor. Cells are washed by spinning the plates, flickingthe contents out of the wells, and applying 150 μl of fresh media toeach well. After a 24 hr coincubation period, the supernatants areharvested and IFN-γsecretion is quantified by ELISA. Wells with thehighest antigen-specific IFN-γ secretion are identified for furtheranalyses of transduced IL-2 gene by PCR.

Identification of Clones Containing Transduced UL-2 gene:

On day 15 after plating, 1×10⁴ cells from each of clones with highestantigen-specific IFN-γ secretion are subjected to direct DNA PCRanalyses with a primer pair of T3 and IL-2 primer 2. T3 primer isspecific for retroviral sequences and L-2 primer 2 contains the sequenceof the 3′ end of the IL-2 gene. Only transduced IL-2 gene will beamplified. Clones that are positive for the transduced EL-2 gene will bechosen for further expansion by REP.

“REP” Expansion of CTL Clones to Therapeutic Numbers:

Each IL-2 transduced tumor-reactive clone is expanded using a singleRapid Expansion Protocol (REP) (Ridell et al., J. Immunol. Methods128:189 (1990)), then re-tested for activity and specificity. In thefirst REP, each clonal well is assumed to have approximately 1×10⁵ cellsand all cells are used.

Components for the REP mix:

Component 25 cm² flask 150 cm² flask viable CTL   1 × 10⁵ 1 × 10⁶Allogeneic PBMCs 2.5 × 10⁷ 2 × 10⁸ OKT3 30 ng/ml 30 ng/ml CM 25 ml 75 mlAIM V 75 ml

On day 0 PBMCs are thawed, washed twice, resuspended in CM andirradiated (34 Gy) as described above. PBMC and OKT3 are added to CM,mixed well, and aliquots are transferred to tissue culture flasks.Viable cells are added last. Flasks are incubated upright at 37° C. in5% CO₂.

On day 2 IL-2 is added to 50 CU/ml.

On day 5, 20 ml (130 ml for a 175 cm² flask) of culture supernatant areremoved by aspiration (cells are retained on the bottom of the flask).Media are replaced with CM containing 50 CU/ml IL-2.

On day 8 an aliquot of cells is removed for counting and re-assay. Cellsare assayed for peptide specificity and tumor recognition bycoincubation assay and ELISA. If cell density is greater than 1×10⁶/ml,cells are split into additional flasks or transferred to Baxter 3 literculture bags. IL-2 is added to 50 CU/ml. Fungizone is added to 1.25mcg/ml and 1 ml/l Cipro is added.

On day 9, the most active clones are tested for their viability afterIL-2 withdrawal. Cells (2×10⁵) from each clone are washed with CM twice,resuspended in 200 μl CM and plated in wells of 96-well flat-bottomplate. On day 13, cells are assayed for their viability using[3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] (MTS) assay. The clones with the highest viability arechosen for therapeutic infusion.

On day 11, IL-2 is added to REP flasks at 50 CU/ml. Cells are split ifdensity exceeds 1.5×10⁶ cells/ml.

The most active IL-2 transduced clones are expanded further totherapeutic numbers with additional REP cycles. In these subsequent REPcycles, CTL are counted and the specified number is used (above Table).In the REP cycle immediately preceding infusion, Fungizone and Cipro areadded on day 8, and AIM V media is used.

Preparation of the Final Product:

On day 14-20 the final product is prepared for patient infusion. Thecontents (cells and media) of flasks are transferred to 250 mlcentrifuge tubes, while cells in Baxter culture bags are usuallyharvested using a Baxter/Fenwal continuous centrifuge cell harvestersystem. Aliquots will be taken from representative bags and pooled for agram test. Cells will be spun to pellet (1,000 rpm, 15 min, R/T) andcombined in a single tube, then washed by resuspension in 0.9% sodiumchloride followed by centrifugation, and finally resuspended in 45-150ml of 0.9% sodium chloride. Human albumin (25%) is added to a finalconcentration of 2.5%. Aliquots are removed for cell count and viabilitytesting by trypan blue exclusion, and for quality control testing. Thefinal product is then infused intravenously as soon as possible.

Example 3

This example describes the generation of melanoma-reactive,IL-2-transduced TILs.

Multiple TILs cultures derived from tumor fragments and/or digests areset up according to the procedures described in Example 2. TILs aretransduced with SBIL-2 retroviral vector with or without REP, dependingon their growth rates. A rapidly growing TILs culture is transducedwithout REP, whereas a slowly growing TILs culture needs a “transductionREP” to stimulate TILs for transduction. All SBIL-2-transduced TILs willundergo a REP to be expanded to the therapeutic number (treatment REP)before they are infused to autologous patients.

Transduction of a Rapidly Growing TIL Culture with SBIL-2 RetroviralVector:

All transductions are performed in the wells of 24-well plates. Wellsare pre-coated with Retronectin followed by SBIL-2 retroviral vector.Retronectin is coated as follows: 2 mls of 1×PBS containing 50 μg/mlretronectin are placed in each well of a 24-well plate at 4° C.overnight or at room temperature for 2 hours. Wells are blocked with 2mls of 1% human albumin in 1×PBS at room temperature for 30 minutes, andwashed once with 2 mls of 1×PBS containing 2.5% HEPES, pH 7.0 (v/v).SBIL-2 retroviral supernatant (2 ml) will be applied to eachRetronectin-coated well and incubated at 32° C. for 2 hours followed by4-16 hours at 4° C. Retroviral supernatant will be removed and 0.5 to1×10⁶ TILs in CM supplemented with 1,000 CU/ml IL-2 will be applied ontoRetronectin and retroviral vector-coated wells. The culture is incubatedat 37° C., 5% CO₂ in an incubator overnight. This procedure is repeateddaily for four days by transferring the TILs from previous transductionwells to wells freshly coated with SBIL-2 retroviral vector. At the endof transduction, cells are washed and maintained in CM supplemented with1,000 CU/ml IL-2.

Transduction of Slowly Growing TILs Cultures with SBIL-2 RetroviralVector (Transduction REP):

Rapid Expansion Protocol (REP):

TILs are expanded in the presence of OKT3 and allogenic feeder PBMCsusing an REP as approved by the FDA for use at the Fred HutchinsonCancer Center (their protocol # 107.00) and at the NCI in protocols98-C-95 and 99-C-158. In this REP protocol, TILs are stimulated withOKT3 (ORTHOCLONE OKT3®, obtained from commercial sources), irradiatedallogeneic feeder cells and IL-2. All components are mixed together in atissue culture flask, then TILs are added.

Component 25 cm² flask 175 cm² flask Viable TILs   1 × 10⁵ 1 × 10⁶allogeneic PBMCs 2.5 × 10⁷ 2 × 10⁸ OKT3 30 ng/ml 30 ng/ml CM 25 ml 75 mlAIM V 75 ml

Allogeneic PBMCs are from frozen vials obtained from harvested PBMCs. Onday 0, PBMCs are thawed, washed and resuspended in CM, and irradiated.Irradiation of allogeneic cells is performed in an MS Nordion Gammacell1000, Model 38.3 irradiator, using 5000 rads from a Cs137 source.Testing includes a control culture flask containing only irradiatedcells, to verify that they have not proliferated.

On day 2 IL-2 is added to 1,000 CU/ml.

On day 5, 20 ml (130 ml for a 175 cm² flask) of culture supernatant areremoved by aspiration (cells are retained on the bottom of the flask).Media are replaced with CM (50% CM/50% AIM V for a 175 cm² flask)containing 1,000 CU/ml IL-2.

Transduction of TIL cells with SBIL-2:

On day 7 after the onset of a REP for TILs, the transduction ofreplicates of 1×10⁶ cells are performed in the wells of 6-well plates.Wells are precoated with Retronectin followed by SBIL-2 retroviralvector. Retronectin is coated as follows: 2 mls of 1×PBS containing 50μg/ml Retronectin is placed in each well of a 6-well plate at 4° C.overnight or at room temperature for 2 hours. Wells are blocked with 2mls of 1% human albumin in 1×PBS at room temperature for 30 minutes andwashed once with 2 mls of 1×PBS containing 2.5% HEPES, pH 7.0 (v/v).SBIL-2 retroviral supernatant (8 ml) is applied to eachRetronectin-coated well and incubated at 32° C. for 2 hours followed by4-24 hours at 4 (C. Retroviral supernatant is removed and 1×10⁶ TILcells in CM-supplemented with 1,000 CU/ml IL-2 are applied ontoRetronectin and retroviral vector-coated wells. The culture is incubatedat 37° C., 5% CO₂ overnight. This procedure is repeated on day 8 bytransferring the TILs culture from previous transduction wells to thewells freshly coated with SBIL-2 retroviral vector. On day 9, cells arewashed and maintained in CM supplemented with IL-2 in a new tissueculture flask. If the cell density increases above 2×10⁶ cells/ml, cellsare split to half of the density and fed within an equal volume of CMplus 1,000 CU/ml IL-2.

Gibbon Ape Leukemia Virus (GALV) Envelope Gene-PCR and S⁺/L⁻ Assays forReplication-Competent Retrovirus (RCR):

SBIL-2-transduced TILs are sent to National Gene Vector Laboratory NGVL)for GALV envelope gene-PCR and S+/L assays to test replication-competentretrovirus (RCR) according to their published methods (Chen et al.,Human Gene Therapy 12: 61-70 (2001)). If PCR results are positive andthe patient has not been started on chemotherapy, that patient is nottreated. In case of a positive RCR PCR for SBIL-2-transduced TIL cellsfrom a patient, who has already received chemotherapy, SBIL-2-transducedTIL cells are not administered. Instead, peripheral blood stem cellscollected from the aphereses after G-CSF mobilization prior to thechemotherapy are infused.

Anti-melanoma Activity Assay for the SBIL-2-Transduced and UntransducedTIL Cells:

Briefly, 1×10⁵ effector cells per well of a flat-bottom 96-well tissueculture plate are coincubated with 1×10⁵ target cells. Typically, 6 to 8different target cells are tested, including T2 cells pulsed with 1.0μg/ml gp100:280-288 or gp100:209-217 peptide or MART 1:27-35 peptide,two HLA-mismatched melanoma cell lines and two HLA-A2 matched melanomacell lines, and patients' autologous tumor cell lines or frozen tumorcells. After 24 hours of incubation, supernatants are harvested andIFN-γ is quantified by ELISA capture assay. The transduced bulk TILscultures, which release 200 pg/ml IFN-γ against the autologous tumor orfrozen tumor cells, are further expanded.

Detection of the Tranduced SBIL-2 Gene and its Transcript:

Genomic DNA PCR is used to detect the transduced genetic material. Acell pellet of 10⁵ cells (100,000) is resuspended in 1,000 μl ofQuickExtract™DNA extraction solution 1.0. Ten microliters of thissolution containing 1,000-cell-equivalent DNA are subjected to PCRreaction containing IL-2 Primer 1 (5′GGAGGCCTGGATCCATGTACAGGATGCAACTCCT3′ [SEQ ID NO: 1]) and Primer 17 (5′CTTCTTGGGCATGTAAAACT 3′ [SEQ ID NO:2]). The PCR products are fractionated on a 2% agarose gel. The presenceof a 221-bp fragment, not present in untransduced cells, is defined aspositive.

The transduced IL-2 gene expression is evaluated by RT-PCR. Transducedcells (1×10⁶) are used for RNA extraction using STRATAGENE AbsolutelyRNA™ RT-PCR Miniprep Kit. The total RNA is subjected to firs-strand cDNAsynthesis by Amersham Pharmacia Biotech Kit. The first-strand cDNA issubjected to PCR reaction containing Primer 16(5′GTCAGCGGGGGGTCTTTCATT3′ [SEQ ID NO: 3]) and Primer 2(5′GGGTCGACGGATCCTCAAGTTAGGTTGAGATGA3′ [SEQ ID NO: 4]). The PCR productsare fractionated on a 1% agarose gel. The presence of an 885-bpfragment, not present in untransduced cells, is defined as positive forSBIL-2 vector-derived IL-2 mRNA transcript.

“REP” Expansion of SBIL-2 Transduced TILs to Therapeutic Numbers:

SBIL-2-transduced TILs are expanded using a single REP, then re-testedfor activity and specificity according to the protocol as describedabove. Eight days prior to cell harvest and re-infusion, an aliquot ofcells is removed for counting and re-assay. Cells are assayed forpeptide specificity and tumor recognition by co-incubation assay andELISA as described above. If cell density is greater than 1×10⁶/ml,cells are split into additional flasks or transferred to Baxter 3 literculture bags. IL-2 is added to 1,000 CU/ml. Fungizone is added to 1.25mcg/ml and Cipro is added to 5-10 mcg/ml. On day 11, IL-2 is added toREP flasks at 1,000 CU/ml. Cell cultures are split as needed.

On day 14, cells are harvested and either prepared for additional REPcycles or cryopreserved. If cells have grown to sufficient numbers forpatient treatment, a sample is collected from each flask formicrobiology tests 2-3 days before the beginning of TIL therapy (thetest takes 2 days). IL-2 is added to 1,000 CU/ml on day 14 and every 3days until the final product is prepared for infusion.

Preparation and Testing of the Final Product:

On day 12-20 the final product is prepared for patient infusion. Thecontents (cells and media) of flasks are transferred to 250 mlcentrifuge tubes, while cells in Baxter culture bags are usuallyharvested using a Baxter/Fenwal continuous centrifuge cell harvestersystem. Aliquots are taken from representative bags and pooled for agram test. Cells are spun to pellet (1,000 rpm, 15 min, RJT) andcombined in a single tube, then washed by resuspension in 0.9% sodiumchloride, followed by centrifugation, and finally resuspended in 45-400ml of 0.9% sodium chloride. Human albumin (25%) is added to a finalconcentration of 2.5%. Aliquots are removed for cell count and viabilitytesting by trypan blue exclusion, and for quality control testing. Thefinal product is infused intravenously as soon as possible.

All references; including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference aid were set forth in its entiretyherein.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, aid the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of preparing autologous T-lymphocytes for re-introductioninto a patient having cancer, which method comprises: (i) obtainingperipheral blood mononuclear cells (PBMCs) from a patient immunized withan antigen of the cancer, (ii) stimulating the PBMCs with the antigen ofthe cancer in vitro, and (iii) transducing the PBMCs with a retroviralvector, which (a) comprises and expresses a human interleukin-2 (IL-2)coding sequence operably linked to a retroviral promoter, (b) does notcomprise an exogenously introduced gene that enables phenotypicselection, and (c) comprises a viral envelope that efficientlytransduces CD8+ T-lymphocytes, whereupon autologous T-lymphocytes areprepared for re-introduction into a patient having cancer.
 2. The methodof claim 1, wherein the cancer is melanoma.
 3. The method of claim 2,wherein the antigen of the cancer is gp100.
 4. The method of claim 3,wherein the antigen is the 209-2M peptide (SEQ ID NO: 5).
 5. The methodof claim 1, wherein the cancer is breast cancer.
 6. The method of claim5, wherein the antigen of the cancer is Her-2/Neu.
 7. The method ofclaim 1, wherein the cancer is prostate cancer.
 8. The method of claim7, wherein the antigen of the cancer is prostate-specific antigen (PSA).9. The method of claim 1, wherein the cancer is colon cancer.
 10. Themethod of claim 9, wherein the antigen of the cancer is carcinoembryonicantigen (CEA).
 11. The method of claim 1, wherein the viral envelopeprotein is Gibbon ape leukemia virus envelope (GALV).
 12. The method ofclaim 1, wherein the retroviral vector further comprises and expresses ahuman IL-2 receptor α-chain coding sequence.
 13. The method of claim 1,wherein the method further comprises introducing into the PBMCs a vectorcomprising and expressing a human IL-2 receptor α-chain coding sequenceoperably linked to a promoter.